Carbohydrate components of woody biomass (cellulose and hemicellulose) represent an abundant potential source of sugars for microbial conversion into renewable fuels, plastics, and other chemicals (7, 18, 35). However, cost-effective depolymerization of this complex material to produce fermentable sugar streams remains a major challenge (3, 35). Pretreatment processes such as dilute mineral acids at elevated temperature and pressures open the structure of woody biomass to increase the effectiveness of cellulase enzymes, and hydrolyze the pentose polymers of hemicellulose into monomers. Unwanted side reactions from this pretreatment also produce a mixture of compounds (furans, acetate, soluble products from lignin, and others) that inhibit growth and retard fermentation (1, 18, 31). Most inhibitors can be removed or neutralized by separating the solubilized sugars from the cellulose-enriched fiber using counter-current washing followed by over-liming (25, 26). However, these added process steps would also add cost to renewable products. By developing robust biocatalysts that are resistant to side products from pretreatment it should be possible to design a simpler process (13, 14).
Furfural, the dehydration product of xylose, is of particular importance as a fermentation inhibitor in hemicellulose hydrolysates (1, 31). Furfural concentrations in hemicellulose hydrolysates have been correlated with toxicity (39). The addition of furfural to over-limed hemicellulose hydrolysates has been shown to restore toxicity (25, 26). In model studies with various hydrolysate inhibitors, furfural was unique in potentiating the toxicity of other compounds (39). Furan alcohols (reduced products) are less toxic than the respective aldehydes (38, 39). Several genes encoding oxidoreductases that reduce furfural and 5-hydroxymethylfurfural (5-HMF; dehydration product of hexose sugars) have been implicated in furan tolerance in Saccharomyces cerevisiae (2, 20, 22, 23) and in E. coli (28-30, 37).
Furfural-resistant mutants of ethanologenic Escherichia coli have been isolated and characterized (28, 29, 37). Resistance to low concentrations of furfural was found to result from the silencing of yqhD, an NADPH-dependent, furfural oxidoreductase that is induced by furfural (28, 29, 37). Although there are multiple NADPH-furfural reductases in E. coli and conversion of furfural to the less toxic alcohol would be generally regarded as beneficial, the unusually low Km of YqhD for NADPH appears to compete with biosynthesis for NADPH (29). Metabolic routes for the anaerobic production of NADPH during xylose fermentation are quite limited (12, 16, 34). The metabolism of furfural by YqhD is proposed to inhibit growth and fermentation by depleting the pool of NADPH below that required for essential biosynthetic reactions (28, 29, 37). Sulfate assimilation was identified as a site that is particularly sensitive to NADPH limitation (28). Furan toxicity (furfural and 5-HMF) can be minimized by a variety of approaches that increased the availability of NADPH (FIG. 1) (28-30).
NADH is abundant during fermentation and represents a preferred reductant for furfural conversion to the less toxic alcohol, eliminating any burden on the NADPH pool. Our laboratory previously cloned the E. coli fucO gene (11), an NADH-dependent, L-1,2 propanediol reductase that is induced during fucose catabolism (8, 10).